Project Summary With the entry into a new era of gene editing, and especially the availability of CRISPR/Cas9 tools, targeted genomic modifications of the rabbit (Oryctolagus cuniculus) have now become a scientific reality. The rabbit is the classic and primary antibody producer thanks to its distinct immune biology (e.g., gene conversion). In recent years, heavy chain-only antibodies (HCAbs), originally discovered in the camelids, with superior single chain-based antigen binding and physico-chemical properties (compared to conventional antibodies) have emerged as the latest class of next-generation biologics for diagnostic and therapeutic purposes ? with the first drugs awaiting approval. It is in this context that we propose (Phase I) to genetically modify the rabbit immune system towards the endogenous production of HCAb-type binders. Through preliminary work we have generated HCAb founder rabbits. In this project, we will demonstrate downstream feasibility towards isolation of single chain monoclonal HCAbs in response to immunization with a standard immunogen. In combination with a confirmation of germline transmission, we intend to achieve an initial proof of concept for our HCAb rabbit model to establish it as a breedable line. In our Aim 1, we focus on Basic Validation/Expansion of Recombinant Rabbits for the Production of HCAbs by verification of genome editing outcomes, HCAb expression and breeding. In Aim 2, we focus on Basic Characterization of HCAb Model Rabbits & Downstream Detection of HCAbs by identifying expression of common wildtype isotypes and by screening of immune libraries for HCAbs with subsequent detection of high-affinity monoclonal binders. We envision our HCAb rabbit platform to translate into the following advantages: (a) Predominant HCAb-IgG isotype responses (in contrast to camelids), (b) Binding domain diversification by gene conversion (typical for rabbits), (c) Enhanced access to HCAbs due to advantageous breeding characteristics and defined experimental procedures, as well as access to immune cells from bone marrow, spleen, and thymus that only a small animal can provide (in contrast to camels), (d) A more user-friendly/more available resource for HCAbs, (e) In vivo affinity matured HCAbs from which monoclonal mHCAbs can be detected, (f) Genetic access to enable further modifications to create rabbit lines with alternative immunological, physiological features (changes to HCAb binding domains, enabling of isotype switching, introduction of transgenes, etc.). In Phase II, based on the progressing interest from biotech, diagnostic, and therapeutic fields and due to the intrinsic properties and adaptability of HCAbs, we will work to further improve the model and to utilize this novel platform for the discovery of clinically significant HCAbs.